Mutations in the P-type cation-transporter ATPase 4, PfATP4, mediate resistance to both aminopyrazole and spiroindolone antimalarials.

Aminopyrazoles are a new class of antimalarial compounds identified in a cellular antiparasitic screen with potent activity against Plasmodium falciparum asexual and sexual stage parasites. To investigate their unknown mechanism of action and thus identify their target, we cultured parasites in the presence of a representative member of the aminopyrazole series, GNF-Pf4492, to select for resistance. Whole genome sequencing of three resistant lines showed that each had acquired independent mutations in a P-type cation-transporter ATPase, PfATP4 (PF3D7_1211900), a protein implicated as the novel Plasmodium spp. target of another, structurally unrelated, class of antimalarials called the spiroindolones and characterized as an important sodium transporter of the cell. Similarly to the spiroindolones, GNF-Pf4492 blocks parasite transmission to mosquitoes and disrupts intracellular sodium homeostasis. Our data demonstrate that PfATP4 plays a critical role in cellular processes, can be inhibited by two distinct antimalarial pharmacophores, and supports the recent observations that PfATP4 is a critical antimalarial target.

Targeting Plasmodium PI(4)K to eliminate malaria.

Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria.

A high-throughput screen to identify inhibitors of ATP homeostasis in non-replicating Mycobacterium tuberculosis.

Growing evidence suggests that the presence of a subpopulation of hypoxic non-replicating, phenotypically drug-tolerant mycobacteria is responsible for the prolonged duration of tuberculosis treatment. The discovery of new antitubercular agents active against this subpopulation may help in developing new strategies to shorten the time of tuberculosis therapy. Recently, the maintenance of a low level of bacterial respiration was shown to be a point of metabolic vulnerability in Mycobacterium tuberculosis. Here, we describe the development of a hypoxic model to identify compounds targeting mycobacterial respiratory functions and ATP homeostasis in whole mycobacteria. The model was adapted to 1,536-well plate format and successfully used to screen over 600,000 compounds. Approximately 800 compounds were confirmed to reduce intracellular ATP levels in a dose-dependent manner in Mycobacterium bovis BCG. One hundred and forty non-cytotoxic compounds with activity against hypoxic non-replicating M. tuberculosis were further validated. The resulting collection of compounds that disrupt ATP homeostasis in M. tuberculosis represents a valuable resource to decipher the biology of persistent mycobacteria.

Imaging of Plasmodium liver stages to drive next-generation antimalarial drug discovery.

Most malaria drug development focuses on parasite stages detected in red blood cells, even though, to achieve eradication, next-generation drugs active against both erythrocytic and exo-erythrocytic forms would be preferable. We applied a multifactorial approach to a set of >4000 commercially available compounds with previously demonstrated blood-stage activity (median inhibitory concentration < 1 micromolar) and identified chemical scaffolds with potent activity against both forms. From this screen, we identified an imidazolopiperazine scaffold series that was highly enriched among compounds active against Plasmodium liver stages. The orally bioavailable lead imidazolopiperazine confers complete causal prophylactic protection (15 milligrams/kilogram) in rodent models of malaria and shows potent in vivo blood-stage therapeutic activity. The open-source chemical tools resulting from our effort provide starting points for future drug discovery programs, as well as opportunities for researchers to investigate the biology of exo-erythrocytic forms.

Identification of inhibitors for putative malaria drug targets among novel antimalarial compounds.

The efficacy of most marketed antimalarial drugs has been compromised by evolution of parasite resistance, underscoring an urgent need to find new drugs with new mechanisms of action. We have taken a high-throughput approach toward identifying novel antimalarial chemical inhibitors of prioritized drug targets for Plasmodium falciparum, excluding targets which are inhibited by currently used drugs. A screen of commercially available libraries identified 5655 low molecular weight compounds that inhibit growth of P. falciparum cultures with EC(50) values below 1.25µM. These compounds were then tested in 384- or 1536-well biochemical assays for activity against nine Plasmodium enzymes: adenylosuccinate synthetase (AdSS), choline kinase (CK), deoxyuridine triphosphate nucleotidohydrolase (dUTPase), glutamate dehydrogenase (GDH), guanylate kinase (GK), N-myristoyltransferase (NMT), orotidine 5'-monophosphate decarboxylase (OMPDC), farnesyl pyrophosphate synthase (FPPS) and S-adenosylhomocysteine hydrolase (SAHH). These enzymes were selected using TDRtargets.org, and are believed to have excellent potential as drug targets based on criteria such as their likely essentiality, druggability, and amenability to high-throughput biochemical screening. Six of these targets were inhibited by one or more of the antimalarial scaffolds and may have potential use in drug development, further target validation studies and exploration of P. falciparum biochemistry and biology.

A chemical genetic screen in Mycobacterium tuberculosis identifies carbon-source-dependent growth inhibitors devoid of in vivo efficacy.

Candidate antibacterials are usually identified on the basis of their in vitro activity. However, the apparent inhibitory activity of new leads can be misleading because most culture media do not reproduce an environment relevant to infection in vivo. In this study, while screening for novel anti-tuberculars, we uncovered how carbon metabolism can affect antimicrobial activity. Novel pyrimidine-imidazoles (PIs) were identified in a whole-cell screen against Mycobacterium tuberculosis. Lead optimization generated in vitro potent derivatives with desirable pharmacokinetic properties, yet without in vivo efficacy. Mechanism of action studies linked the PI activity to glycerol metabolism, which is not relevant for M. tuberculosis during infection. PIs induced self-poisoning of M. tuberculosis by promoting the accumulation of glycerol phosphate and rapid ATP depletion. This study underlines the importance of understanding central bacterial metabolism in vivo and of developing predictive in vitro culture conditions as a prerequisite for the rational discovery of new antibiotics.

Identification of novel therapeutic targets for HIV infection through functional genomic cDNA screening.

Despite decades of research, HIV remains a global health threat. Issues of multi-drug resistance and lack of an effective vaccine have recently led to the targeting of host factors for anti-viral drug development. While a few genome-wide screens for novel HIV co-factors have been reported, the promise of finding a therapeutic target has yet to be realized. Here, we report a screen of a cDNA library representing 15,000 unique genes in an infectious HIV system, and show that genomic screening can lead to the identification of novel proviral host factors. Mixed lineage kinase 3 (MLK3/MAP3K11) was identified as one of the strongest enhancers of infection and mutant studies show that its activity is dependent on its kinase function. Consistent with its known role in the activation of the AP-1 pathway through JNK kinase, MLK3 was able to enhance Tat-dependent HIV transcription in vitro thus leading to an increase in infection signal. RNA interference studies confirm the involvement of endogenous MLK3 in HIV infection, further implicating this kinase as a potential therapeutic target.

Homogeneous high-throughput screening assays for HIV-1 integrase 3beta-processing and strand transfer activities.

HIV-1 integrase (HIV-IN) is a well-validated antiviral drug target catalyzing a multistep reaction to incorporate the HIV-1 provirus into the genome of the host cell. Small molecule inhibitors of HIV-1 integrase that specifically target the strand transfer step have demonstrated efficacy in the suppression of virus propagation. However, only few specific strand transfer inhibitors have been identified to date, and the need to screen for novel compound scaffolds persists. Here, the authors describe 2 homogeneous time-resolved fluorescent resonance energy transfer-based assays for the measurement of HIV-1 integrase 3'-processing and strand transfer activities. Both assays were optimized for high-throughput screening formats, and a diverse library containing more than 1 million compounds was screened in 1536-well plates for HIV-IN strand transfer inhibitors. As a result, compounds were found that selectively affect the enzymatic strand transfer reaction over 3beta processing. Moreover, several bioactive molecules were identified that inhibited HIV-1 reporter virus infection in cellular model systems. In conclusion, the assays presented herein have proven their utility for the identification of mechanistically interesting and biologically active inhibitors of HIV-1 integrase that hold potential for further development into potent antiviral drugs.

Identification of cellular cofactors for human immunodeficiency virus replication via a ribozyme-based genomics approach.

Ribozymes are small, catalytic RNA molecules that can be engineered to down-regulate gene expression by cleaving specific mRNA. Here we report the selection of hairpin ribozymes that inhibit human immunodeficiency virus (HIV) replication from a combinatorial ribozyme library. We identified a total of 17 effective ribozymes, each capable of inhibiting HIV infection of human CD4(+) cells. These ribozymes target diverse steps of the viral replication cycle, ranging from entry to transcription. One ribozyme suppressed HIV integration and transcription by inhibiting the expression of the Ku80 subunit of the DNA-activated protein kinase. Another ribozyme specifically inhibited long terminal repeat transactivation, while two additional ones blocked a step that can be bypassed by vesicular stomatitis virus G-protein pseudotyping. The function of Ku80 in HIV replication and its mechanism of action were further confirmed using short interfering RNA. Identification of the gene targets of these and other selected ribozymes may reveal novel therapeutic targets for combating HIV infection.